Our aim is to explore basic aspects of human RBC membrane function, thereby providing information which in turn could be useful in understanding and managing RBC disorders and in prolonging the shelf life of blood stored for transfusion. Observation of endocytosis in RBC and ghosts provides opportunities for studying membrane invagination, constriction, fusion and detachment. In white ghost endocytosis spectrin clustering will be studied using transmission electron microscopy and ferritin-labelled anti-spectrin antibody, while alkaline phosphatase attack will be used to study the requirements of endocytosis for protein phosphorylation. In red ghost endocytosis the vacuoles will be made heavier by tagging them with cationized ferritin. Then after isolation the vacuole content of spectrin and Ca, Mg-ATPase will be measured in order to determine their role in endocytosis. The role of Ca stimulated transglutaminase in RBC and red ghost endocytosis will be tested using the inhibitor, histamine. 3H-vinblastine will be used to study the entry and distribution of this drug into RBC during endocytosis. 45Ca movement within RBC will be studied to identify the 45Ca membrane binding sites in normal circumstances, after exposure to endocytosis inducing drugs, and in disease like sickle cell anemia. We will search for potentially functional microenvironments in hypotonic and isotonic disrupted white ghosts using the enzymic markers acetylcholinesterase, NADH oxidoreductase, glyceraldehyde phosphate dehydrogenase, Mg-ATPase and Na, K-ATPase. We will attempt to purify the myosif-like actin-activated Mg-ATPase and study its mechanism of action in normal and abnormal RBC. Defective primaquine endocytosis not responding to brief restoration of ATP occurs after prolonged RBC storage, in parallel with the appearance of membrane protein complexes which may represent oxidative attack, or the effects of Ca. The extent and type of these complexes will be studied by two-dimensional gel electrophoresis, and their appearance will be correlated with the impairment or primaquine endocytosis and post-transfusion RBC survival. We plan to study the sites of binding for the increased membrane 45Ca seen in sickle cell anemia, spectrin function in white ghost endocytosis in hereditary pyropoikilocytosis and the mechanism by which dimethyl adipimidate restor (Text Truncated - Exceeds Capacity)